T cell-associated immunity induced by heterologous recombinant BCG and purified protein vaccination confers cross-variant protection against SARS-CoV-2.

Previously, we have developed a recombinant BCG-based vaccine (rBCG) expressing a chimeric protein with spike and nucleocapsid epitopes (rChimera) to combine BCG's innate immune training capacity with SARS-CoV-2-specific adaptive immune responses. The heterologous prime-boost rBCG/rChimera+Alum regimen induced strong humoral and cellular immunity, conferring protection against the ancestral Wuhan strain in K18-hACE2 mice. Here, we used knockout mice lacking functional B cells, CD4+, or CD8+ T lymphocytes expressing hACE2 to uncover the mechanisms of anti-viral immune protection, while also evaluating vaccine efficacy against JN.1. Vaccination induced high titers of anti-rChimera antibodies with modest neutralizing capacity against the Wuhan SARS-CoV-2 strain. Nonetheless, vaccinated B lymphocyte KO mice still showed reduced viral loads, suggesting that humoral immunity may not represent the predominant protective mechanism in this model. Vaccinated mice displayed a strong Th1-biased cellular profile characterized by increased IFN-γ production and multifunctional CD4+ T cell responses, alongside activated CD8+ T cells responses. CD4+ and CD8+ T cell-deficient animals exhibited loss of vaccine-associated protection, supporting an important contribution of T cells to viral control. Additionally, immunized IFN-γ knockout mice revealed only partial protection, suggesting that IFN-γ contributes to, but is not strictly required, for vaccine-induced immunity. We also observed that macrophages derived from vaccinated animals displayed enhanced inflammatory responsiveness following heterologous stimulation. Remarkably, vaccination conferred cross-protection against JN.1, despite lack of neutralization antibodies. These findings indicate that rBCG/rChimera+Alum vaccination induces an integrated innate and predominant T cell protective immunity cross-reactive with JN.1, supporting the rBCG-based platforms as a promising approach for COVID-19 vaccine development.